Biochemical characterization of RssA-RssB, a two-component signal transduction system regulating swarming behavior in Serratia marcescens

Our previous study had identified a pair of potential two-component signal transduction proteins, RssA-RssB, involved in the regulation of Serratia marcescens swarming. When mutated, both rssA and rssB mutants showed precocious swarming phenotypes on LB swarming agar, whereby swarming not only occurred at 37 degrees C but also initiated on a surface of higher agar concentration and more rapidly than did the parent strain at 30 degrees C. In this study, we further show that the predicted sensor kinase RssA and the response regulator RssB bear characteristics of components of the phosphorelay signaling system. In vitro phosphorylation and site-directed mutagenesis assays showed that phosphorylated RssA transfers the phosphate group to RssB and that histidine 248 and aspartate 51 are essential amino acid residues involved in the phosphotransfer reactions in RssA and RssB, respectively. Accordingly, while wild-type rssA could, the mutated rssA(H248A) in trans could not complement the precocious swarming phenotype of the rssA mutant. Although RssA-RssB regulates expressions of shlA and ygfF of S. marcescens (ygfF(Sm)), in vitro DNA-binding assays showed that the phosphorylated RssB did not bind directly to the promoter regions of these two genes but bound to its own rssB promoter. Subsequent assays located the RssB binding site within a 63-bp rssB promoter DNA region and confirmed a direct negative autoregulation of the RssA-RssB signaling pathway. These results suggest that when activated, RssA-RssB acts as a negative regulator for controlling the initiation of S. marcescens swarming.     

Clostridium difficile toxin A regulates inducible cyclooxygenase-2 and prostaglandin E2 synthesis in colonocytes via reactive oxygen species and activation of p38 MAPK

Clostridium difficile toxin A induces acute colitis with neutrophil infiltration and up-regulation of numerous pro-inflammatory mediators, but the contribution of cyclooxygenase-2 (COX-2) induction in this infection is unknown. We report here that toxin A induces expression of COX-2 and secretion of prostaglandin E2 (PGE2) in a dose- and time-dependent manner in cultured NCM460 human colonocytes and in human intestinal xenografts. This induction was blocked by SB203580, a p38 MAPK inhibitor, which also decreased the phosphorylation of MSK-1, CREB/ATF-1, and COX-2 promoter activity following toxin A stimulation. Gel shift assays indicated that CREB/ATF-1 was the major proteins binding to the COX-2-CRE. Moreover, colonocytes exposed to toxin A produced reactive oxygen species (ROS), which activated p38 MAPK, MSK-1, and CREB/ATF-1, leading to subsequent COX-2 induction and PGE2 secretion. In intact mice, blockage of p38 MAPK inhibited toxin A-mediated induction of COX-2 in enterocytes as well as lamina propria cells, and significantly blocked the toxin A-induced ileal secretion of fluid and PGE2. Furthermore, a selective COX-2 inhibitor also diminished toxin A-associated ileal fluid and PGE2 secretion. The main signaling pathway for toxin A induction of human COX-2 involves ROS-mediated activation of p38 MAPK, MSK-1, CREB, and ATF-1. Toxin A triggers ileal inflammation and secretion of fluid via COX-2 induction and release of PGE2.     

Inhibition of HIV-1 maturation via drug association with the viral Gag protein in immature HIV-1 particles

The small molecule 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) potently inhibits human immunodeficiency virus, type 1 (HIV-1) replication by interfering with proteolytic cleavage of the viral Gag protein at a specific site. Here we have demonstrated that the antiviral mechanism involves the association of DSB with Gag at a 1:1 stoichiometry within immature HIV-1 particles. The binding was specific, as mutations in Gag that confer resistance to DSB inhibited the association, which could be competed by DSB but not by the inactive compound betulinic acid. The addition of DSB to purified immature viral cores inhibited the cleavage of Gag at the CA-SP1 junction in vitro, thus reproducing the effect of the drug when present during maturation of HIV-1 particles. Based on these findings, we propose a model in which a trimer of DSB associates with the CA-SP1 junction of adjacent subunits within the Gag polymer. The model may explain the ability of highly similar compounds to specifically target the seemingly unrelated steps of HIV-1 maturation and virus entry.     

Crystal structure of lipoate-protein ligase A bound with the activated intermediate: insights into interaction with lipoyl domains

Lipoic acid is the covalently attached cofactor of several multi-component enzyme complexes that catalyze key metabolic reactions. Attachment of lipoic acid to the lipoyl-dependent enzymes is catalyzed by lipoate-protein ligases (LPLs). In Escherichia coli, two distinct enzymes lipoate-protein ligase A (LplA) and lipB-encoded lipoyltransferase (LipB) catalyze independent pathways for lipoylation of the target proteins. The reaction catalyzed by LplA occurs in two steps. First, LplA activates exogenously supplied lipoic acid at the expense of ATP to lipoyl-AMP. Next, it transfers the enzyme-bound lipoyl-AMP to the epsilon-amino group of a specific lysine residue of the lipoyl domain to give an amide linkage. To gain insight into the mechanism of action by LplA, we have determined the crystal structure of Thermoplasma acidophilum LplA in three forms: (i) the apo form; (ii) the ATP complex; and (iii) the lipoyl-AMP complex. The overall fold of LplA bears some resemblance to that of the biotinyl protein ligase module of the E. coli biotin holoenzyme synthetase/bio repressor (BirA). Lipoyl-AMP is bound deeply in the bifurcated pocket of LplA and adopts a U-shaped conformation. Only the phosphate group and part of the ribose sugar of lipoyl-AMP are accessible from the bulk solvent through a tunnel-like passage, whereas the rest of the activated intermediate is completely buried inside the active site pocket. This first view of the activated intermediate bound to LplA allowed us to propose a model of the complexes between Ta LplA and lipoyl domains, thus shedding light on the target protein/lysine residue specificity of LplA.     

The interaction of phospholipase C-beta3 with Shank2 regulates mGluR-mediated calcium signal

Phospholipase C-beta isozymes that are activated by G protein-coupled receptors (GPCR) and heterotrimeric G proteins carry a PSD-95/Dlg/ZO-1 (PDZ) domain binding motif at their C terminus. Through interactions with PDZ domains, this motif may endow the PLC-beta isozyme with specific roles in GPCR signaling events that occur in compartmentalized regions of the plasma membrane. In this study, we identified the interaction of PLC-beta3 with Shank2, a PDZ domain-containing multimodular scaffold in the postsynaptic density (PSD). The C terminus of PLC-beta3, but not other PLC-beta isotypes, specifically interacts with the PDZ domain of Shank2. Homer 1b, a Shank-interacting protein that is linked to group I metabotropic glutamate receptors and IP3 receptors, forms a multiple complex with Shank2 and PLC-beta3. Importantly, microinjection of a synthetic peptide specifically mimicking the C terminus of PLC-beta3 markedly reduces the mGluR-mediated intracellular calcium response. These results demonstrate that Shank2 brings PLC-beta3 closer to Homer 1b and constitutes an efficient mGluR-coupled signaling pathway in the PSD region of neuronal synapses.     

Structural chemoproteomics and drug discovery

Our laboratories have developed several technologies to accelerate drug discovery process on the basis of structural chemoproteomics. They include SPS technology for the efficient determination of protein structures, SCP technology for the rapid lead generation and SDF technology for the productive lead optimization. Using these technologies, we could determine many 3D structures of target proteins bound with biologically active chemicals including the structure of phosphodiesterase 5/Viagra complex and obtain highly potent compounds in animal models of obesity, diabetes, cancer and inflammation. In this paper, we will discuss concepts and applications of structural chemoproteomics for drug discovery.     

Effect of adenosine triphosphate on phosphate uptake in renal proximal tubule cells: involvement of PKC and p38 MAPK

ATP has been known to act as an extracellular signal and to be involved in various functions of kidney. Renal proximal tubular reabsorption of phosphate (Pi) contributes to the maintenance of phosphate homeostasis, which is regulated by Na+/Pi cotransporter. However, the effects of ATP on Na+/Pi cotransporters were not elucidated in proximal tubule cells (PTCs). Thus, the effects of ATP on Na+/Pi cotransporter and its related signal pathways are examined in the primary cultured renal PTCs. In the present study, ATP inhibited Pi uptake in a time (> 1 h) and dose (>10(-6)M) dependent manner. ATP-induced inhibition of Pi uptake was correlated with the decrease of type II Na+/Pi cotransporter mRNA. ATP-induced inhibition of Pi uptake may be mediated by P2Y receptor activation, since suramin (non-specific P2 receptor antagonist) and RB-2 (P2Y receptor antagonist) blocked it. ATP-induced inhibition of Pi uptake was blocked by neomycin, U73122 (phospholipase C (PLC) inhibitors), bisindolylmaleimide I, H-7, and staurosporine (protein kinase C (PKC) inhibitors), suggesting the role of PLC/PKC pathway. ATP also increased inositol phosphates (IPs) formation and induced PKC translocation from cytosolic fraction to membrane fraction. In addition, ATP-induced inhibition of Pi uptake was blocked by SB 203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by PD 98059 (a p44/42 MAPK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK, which was not blocked by PKC inhibitor. In conclusion, ATP inhibited Pi uptake via PLC/PKC as well as p38 MAPK in renal PTCs.